Role of Rh (rhesus) complex in K‐Cl cotransport (KCC) regulation in red blood cells (RBCs) of Rh and Rh‐associated glycoprotein (RhAg) knockout (KO) mice

Abstract

The Rh blood group is a macromolecular complex of protein subunits (D and CE), RhAG, a NH3/CO2 gas transporter (PNAS 101:17222,2004; FASEB J 22;1,2008), and accessory proteins (ICAM‐4/LW, CD47 and GPB). In the mild hemolytic human Rhnull disorder, the Rh complex is absent/severely deficient. Altered K fluxes have been found in Rhnull RBC (Blood 48: 457, 1976). A double blind study on male (m) or female (f) wild type (WT), and Rh−/− and Rhag−/− KO mice (Transf Clin Biol 13:164,2006) studied: Total, ouabain (O)‐sensitive Na/K pump, O‐insensitive+bumetanide (B)‐sensitive Na‐K‐2Cl cotransport (NKCC), and O‐ and B‐insensitive KCC ± the thiol alkylant N‐ethylmaleimide (NEM), staurosporine (STP, a kinase inhibitor) and Mg removal by A23187.Results1) Total, Na/K pump, NKCC and basal KCC influx were not different between groups. 2) NEM activated KCC by >100 % (p<0.001) in both m/f Rh−/− and RhAg−/−, compared to WT. KCC activation by both STP, and Mg depletion (except for m Rh−/−), was similar in m Rh−/−, m/f Rhag−/− RBCs and WT.ConclusionThiol modification reveals a Rh‐complex role in KCC regulation, i.e. via protein phosphorylation or protein/protein interactions. However, unlike in human Rhnull RBCs, Na/K pump and passive K‐"leak" fluxes were normal, an apparent difference in the Rh complex‐K transport association between mice and man. Support in part by a WSU School of Medicine Grant.